AU2021402788B2

AU2021402788B2 - Use of pharmaceutical composition for treating non-small cell lung cancer

Info

Publication number: AU2021402788B2
Application number: AU2021402788A
Authority: AU
Inventors: Min-Tsang Hsieh; Mien-Chie Hung; Shin-Hun Juang; Sheng-Chu Kuo; Pei-Chih LEE; Hui-Yi Lin; Chang-Hai TSAI
Original assignee: HUNG MIEN CHIE
Current assignee: HUNG MIEN CHIE
Priority date: 2020-12-17
Filing date: 2021-12-14
Publication date: 2024-11-28
Anticipated expiration: 2041-12-14
Also published as: WO2022127751A1; EP4265256A4; CN117120438A; EP4265256A1; TWI798994B; TW202225136A; KR20230131861A; AU2021402788A9; JP2023554532A; JP7646243B2; CA3205490A1; US20240000742A1; AU2021402788A1

Abstract

A use of a pharmaceutical composition is provided. The pharmaceutical composition contains a diarylheptanoid compound or a pharmaceutically acceptable salt thereof. The diarylheptanoid compound has a structure shown in formula (I). Symbols in formula (I) are as defined in the description, and the pharmaceutical composition can inhibit the growth of lung cancer cells. Therefore, the pharmaceutical composition can be used for preparing a drug for treating lung cancer.

Description

USE OF PHARMACEUTICAL COMPOSITION FOR TREATING NON-SMALL CELLLUNGCANCER BACKGROUND

Technical Field

The present disclosure relates to a use of a pharmaceutical composition.

More particularly, the present disclosure relates to a use of a pharmaceutical

composition including a diarylheptanoid compound or a pharmaceutically

acceptable salt thereof for treating lung cancer.

Description of Related Art

Any reference in this specification to prior art, or matter which is said to be

known, is not to be taken as an acknowledgement or admission that such prior

art or matter forms part of the common general knowledge in the field of invention

to which this specification relates.

Cancer, also known as malignant tumor, is an abnormal proliferation of cells,

and these proliferating cells may invade other parts of the body, which is a

disease caused by abnormal control of cell division and proliferation mechanisms.

There is an increasing trend in the number of people suffering from cancer

worldwide, and about 20% of the cancer population in the world is lung cancer

patients. The 5-year survival rate of lung cancer patients after treatment is still

as low as about 15%, which has been the cancer with the highest death rate in

the world for many years.

According to different biological characteristics, treatment and prognosis, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer (NSCLC). About 85-90% of lung cancers are NSCLC, of which lung adenocarcinoma is the most common type of lung cancer in women and non smoking patients. Treatment for lung cancer often depends on the age of patient, past medical history, current health status, type of cancer cells, and stage of the disease. Generally speaking, small cell lung cancer has the characteristics of rapid division and proliferation, and metastases may occur in a short period of time, so that systemic chemotherapy or radiation therapy is the main treatment. The growth of NSCLC is slower, and the occurrence of io metastasis is also slower, so that the principle of treatment depends on the clinical stage of the disease. The radical treatment of early stage (stage1,11)

NSCLC is still based on complete resection of the tumor by surgery. The

treatment principle is mainly chemical drug therapy or chemical drug combined

with radiation therapy for the locally extended stage (stage Ill) including patients

with malignant pericardium or hydropleural effusion and distant metastases

(stage IV) or patients whose physical condition cannot be surgically removed.

However, it is a thorny problem in the treatment of metastatic or advanced

NSCLC that has undergone chemotherapy and relapsed. Current clinical

studies have confirmed that epidermal growth factor receptor-tyrosine kinase

inhibitors (EGFR-TKIs) can be used as the second-line treatment after the first

line chemotherapy fails. But about 40-80% of NSCLC patients have the EGFR

gene mutation, which overexpresses the epidermal growth factor receptor,

leading to rapid growth, metastasis and drug resistance of cancer. Almost all

patients with the EGFR gene mutation relapse within two years after clinical

treatment with EGFR-TKIs, and no effective drugs are available after relapse so far.

SUMMARY

In view of this, the present disclosure seeks to provide a use of a

pharmaceutical composition, which can be used to manufacture a drug for

treating a lung cancer. The pharmaceutical composition includes a

diarylheptanoid compound or a pharmaceutically acceptable salt thereof, which

can inhibit the growth of the epidermal growth factor receptor-tyrosine kinase

inhibitors resistant non-small cell lung cancer cells. Therefore, the

pharmaceutical composition can be used alone or in combination with clinically

used epidermal growth factor receptor-tyrosine kinase inhibitors to treat a EGFR

gene mutation with epidermal growth factor receptor-tyrosine kinase inhibition

drug-resistant lung cancer.

According to one aspect of the present disclosure is to provide a use of a

pharmaceutical composition in manufacture of a drug for treating a lung cancer,

wherein the pharmaceutical composition includes a diaryheptanoid compound or

a pharmaceutically acceptable salt thereof, and the diaryheptanoid compound

has a structure represented by Formula (1):

O O

RaR Re'

Rb Rb' Re Re

Formula (1),

wherein Ra, Rb, Ra'and Rb'are independently H, C1-C2 alkyl, C1-C3 alkoxy,

OH, or -OC(=O)Rd, wherein Rd is 01-03 alkyl or C1-C3 alkanol; Rc is H, C1-C2

alkyl, C3-C6 unsaturated alkyl or C7-C12 arylalkyl with double or triple bonds;

and Re and Re' are independently H, C1-C6 alkyl or C1-C6 alkoxy.

According to the use of the pharmaceutical composition, wherein at least one

of Ra, Rb, Ra', and Rb' can be -OC(=O)Rd, wherein Rd is 01-03 alkyl or 01-03

alkanol.

According to the use of the pharmaceutical composition, wherein the

diarylheptanoid compound can be interconvertible between keto and enol forms,

when Rc is H.

According to the use of the pharmaceutical composition, wherein the

diarylheptanoid compound can be selected from Compound 1, Compound 21a,

Compound 21b, Compound 22a, Compound 22b, Compound 23a, Compound

23b, Compound 24a, Compound 24b, Compound 25, Compound 26, Compound

27, Compound 31 and Compound 33 having a structure represented by Formula

(II): O OH

R2 R2' R3 R3

Formula (II);

wherein R1, R'are OCH respectively, R2, R2'are H respectively, R3, R3'are

H respectively in Compound 1; R1, R1' are OCH respectively, R2, R2' are OR4

respectively, R3, R3' are H respectively in Compound 21a; R1, R1' are OCH

respectively, R2 is OH, R2' is OR4, R3, R3' are H respectively in Compound 21b;

A

R1, R1' are OCH respectively, R2, R2' are OR5 respectively, R3, R3' are H

respectively in Compound 22a; R1, R1' are OCH3 respectively, R2 is OH, R2' is

OR5, R3, R3'are H respectively in Compound 22b; R1, R'are H respectively, R2,

R2'are OR5 respectively, R3, R3'are H respectively in Compound 23a; R1, R'are

H respectively, R2 is OR, R2'is OH, R3, R3'are H respectively in Compound 23b;

R1, R1' are OR4 respectively, R2, R2' are OCH3 respectively, R3, R3' are H

respectively in Compound 24a; Ri is OR4, R1' is OH, R2, R2' are OCH3

respectively, R3, R3' are H respectively in Compound 24b; Ri is OR5, R1' is OH,

R2, R2'are OCH3 respectively, R3, R3'are H respectively in Compound 25; R1, R1'

are OC2H5 respectively, R2, R2' are OR4 respectively, R3, R3' are H respectively

in Compound 26; R1, R1' are OC2H5 respectively, R2, R2' are OR respectively,

R3, R3'are H respectively in Compound 27; R1, R'are C2H5 respectively, R2, R2'

are OR5 respectively, R3, R3'are C2H5 respectively in Compound 31; and R1, R1'

are OCH3 respectively, R2 is OCH, R2' is OR4, R3, R3' are H respectively in

Compound 33; wherein R4 is a structure represented by Formula (i), and R5 is a

structure represented by Formula (ii):

0 0 OH OH OH OH

Formula (i); Formula (ii).

According to the use of the pharmaceutical composition, wherein the

diarylheptanoid compound is selected from Compound 35a, Compound 35c,

Compound 35d, Compound 35e, Compound 36 and Compound 37 having a

structure represented by Formula (111):

0 0 R6 R6' R8 R8 R7 R7'

Formula (111);

wherein R6, R6' are OCH3 respectively, R7, R7' are OR4 respectively, R8 is

CH3 in Compound 35a; R6, R6' are OCH3 respectively, R7, R7' are OR4

respectively, R8 is benzyl in Compound 35c; R6, R6' are OCH3 respectively, R7,

R7' are OR4 respectively, R8 is propargyl in Compound 35d; R6, R6' are OCH3

respectively, R7, R7' are OR4 respectively, R8 is allyl in Compound 35e; R6, R6'

are H respectively, R7, Rare OR4 respectively, R8 is CH3 in Compound 36; and

R6, R6' are OR4 respectively, R7, R7' are OCH3 respectively, R8 is CH3 in

Compound 37; wherein R4 is a structure represented by Formula (i):

0 OH OH

Formula (i).

According to the use of the pharmaceutical composition, wherein the

diarylheptanoid compound can be selected from: 0 OH 0 0 MeO O - eOOe OO OOe

HO 0 0 OH HO 0 0 OH

HO OH HO OH

0 0 MeO 0~ 0O~0 HO 0 0 OH0 H ):IO O HO 0 00 -IeOH 0HO OH HO' OH

0 0 0 0 HO 0 '4' 0 -lCOH

HO O O OH and H3CO OCH 3

. According to the use of the pharmaceutical composition, wherein the

pharmaceutical composition can further include epidermal growth factor receptor

tyrosine kinase inhibitors (EGFR-TKIs). Preferably, the EGFR-TKIs can be

osimertinib, gefitinib, erlotinib or afatinib.

According to the use of the pharmaceutical composition, wherein the lung

cancer can be a non-small cell lung cancer (NSCLC).

According to the use of the pharmaceutical composition, wherein the lung

cancer can be resistant to epidermal growth factor receptor-tyrosine kinase

inhibitors.

According to one aspect, the present invention provides use of a

pharmaceutical composition in the manufacture of a medicament for the

treatment of a non-small cell lung cancer, wherein the pharmaceutical

composition comprises a diarylheptanoid compound or a pharmaceutically

acceptable salt thereof, and the diarylheptanoid compound is selected from:

MeO 0N m Meo - OMe

HO 0 O OH HO 0 O OH

HO OH HO OH

0 0

0O O HO 0 0 OH HO OH and 0 00 0 0 HO 0 N N N OH

HO K OH H3 CO OCH3

According to one aspect, the present invention provides a method of treating

a non-small cell lung cancer, the method comprising administering to a subject in

need thereof an effective amount of a pharmaceutical composition comprising a

diarylheptanoid compound or a pharmaceutically acceptable salt thereof, wherein

the diarylheptanoid compound is selected from:

MeO 0N M MeO - Me

HO o O OH HO 0 O OH

HO OH HO OH

0 0

O O HO 0 O OH HO OH and 0 0 0 0

HOH HOH H3 CO OCH3

The above summary is intended to provide a simplified summary of the

disclosure to provide the reader with a basic understanding of the disclosure.

The summary is not an exhaustive overview of the disclosure, and it is not

intended to identify key/critical elements of embodiments of the present

disclosure or to delineate the scope of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure can be more fully understood by reading the following

detailed description of the embodiment, with reference made to the

accompanying drawings as follows:

Fig. 1A shows the analysis result of the inhibition of H1650 cell growth by the

Q diarylheptanoid compounds of the present disclosure;

Fig. 1B, Fig. 1C and Fig. 1D show the analysis results of the inhibition of the

growth of the epidermal growth factor receptor-tyrosine kinase inhibitors resistant

non-small cell lung cancer (NSCLC) cells by the diarylheptanoid compounds of

the present disclosure;

Fig. 2A shows the analysis result of the inhibition of tumor growth in the GR6

tumor mice by treating Compound 35d alone;

Fig. 2B shows the analysis result of the inhibition of tumor growth in the GR8

tumor mice by treating Compound 35d alone;

Fig. 2C shows the analysis result of the inhibition of tumor growth in the

HCC827 tumor mice by treating Compound 35d alone;

Fig. 2D shows the statistical chart of body weight changes in the tumor mice

by treating Compound 35d alone;

Fig. 3A shows the analysis result of the inhibition of tumor re-progression in

the GR6 tumor mice by treating a combined treatment of Compound 35d and

osimertinib; and

Fig. 3B shows the statistical chart of body weight changes in the GR6 tumor

mice by treating the combined treatment of Compound 35d and osimertinib.

DETAILED DESCRIPTION

The present disclosure provides a novel use of a pharmaceutical

composition, and the pharmaceutical composition includes a diarylheptanoid

compound or a pharmaceutically acceptable salt thereof, which can be used to manufacture a drug for treating a lung cancer. The diarylheptanoid compound of the present disclosure has a structure represented by Formula (1):

O O

)qr Re Rec Rb 4;Rb' Re Re

Formula (1),

wherein Ra, Rb, Ra'and Rb'are independently H, C1-C2 alkyl, C1-C3 alkoxy,

OH, or -OC(=O)Rd, wherein Rd is 01-03 alkyl or C1-C3 alkanol; Rc is H, C1-C2

alkyl, C3-C6 unsaturated alkyl or C7-C12 arylalkyl with double or triple bonds;

and Re and Re' are independently H, C1-C6 alkyl or C1-C6 alkoxy.

At least one of Ra, Rb, Ra', and Rb' of the diarylheptanoid compound of the

present disclosure can be -OC(=O)Rd, and Rd is 01-03 alkyl or 01-03 alkanol.

In addition, the diarylheptanoid compound can be interconvertible between keto

and enol forms, when Rc is H.

The pharmaceutical composition of the present disclosure can further

include epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs),

which can be combined with the diarylheptanoid compound or a pharmaceutically

acceptable salt thereof. The EGFR-TKIs can be osimertinib, gefitinib, erlotinib

or afatinib. The lung cancer treated by the pharmaceutical composition of the

present disclosure can be non-small cell lung cancer (NSCLC). In addition, the

lung cancer can be resistant to the EGFR-TKIs.

Unless otherwise noted, all terms, symbols or other scientific terms or terms

used in the present disclosure have the meanings that are commonly understood

1n by person having ordinary skill in the art. In some cases, terms with conventional meanings are defined herein for clarity and/or immediate reference, and the definitions incorporated herein should be construed as not necessarily substantial different from the conventional meanings in the art. Many of the techniques and procedures described or referenced herein are well known and routinely used by those skilled in the art. Where appropriate, unless otherwise stated, procedures for the use of commercially available kits and reagents are generally performed according to instructions and/or parameters defined by the manufacturer.

Unless contraindicated or noted otherwise, in these descriptions and

throughout this specification, the terms "a" and "an" mean one or more (that is at

least one). Furthermore, genera are recited as shorthand for a recitation of all

members of the genus; for example, the recitation of C1-C3 alkyl is shorthand for

a recitation of all C1-C3 alkyls including methyl, ethyl, propyl, and isomers

thereof.

The diarylheptanoid compounds disclosed in the present disclosure and the

pharmaceutically acceptable salt thereof can be verified by in vitro experiments,

which can inhibit the growth of the EGFR-TKIs resistant NSCLC cells. It can

further be verified by in vivo experiments that the compound disclosed in the

specification and/or at least one pharmaceutically acceptable salt thereof can be

administered to animals suffering from EGFR-TKIs resistant lung cancer (such

as mouse model), and can obtain therapeutic effects. A positive result in one or

more tests is sufficient to demonstrate the actual utility of the tested compound

and/or salt, and an appropriate dosage range and administration route for animals

(such as humans) can be determined based on test results.

Useful pharmaceutical dosage forms for administering the diarylheptanoid

compound of the present disclosure and the pharmaceutically acceptable salt

thereof include, but are not limited to, hard and soft gelatin capsules, tablets,

parenteral injections and oral suspensions. The dosage administered can

depend on factors including the age of the subject, the health and weight of the

subject, the extent of the disease, the type of concomitant treatment (if any), the

frequency of treatment and the nature of the desired effect. Usually the daily

dose of active ingredient may vary, for example from 0.1 to 2000 mg per day.

io For example, 10-500 mg one or more times per day can be effective to achieve

the desired results.

The same dosage form can generally be used when the diarylheptanoid

compounds of the present disclosure and pharmaceutically acceptable salt

thereof are administered stepwise or in combination with at least one other

therapeutic agent. When drugs are administered in a physical combination, the

dosage form and route of administration should be selected based on the

compatibility of the combined drugs. Therefore, "co-administration" in the

specification should be understood to include the concomitant or sequential

administration of at least two agents, or as a fixed-dose combination of at least

two active ingredients.

The diarylheptanoid compound and the pharmaceutically acceptable salt

thereof in the specification can be used as the active ingredient alone, or

administered in combination with at least one second active ingredient, the

second active ingredient can be selected from, for example, other active

1) ingredients known to be useful in the treatment of patients with NSCLC, in particular the EGFR-TKIs.

The following specific examples are used to further illustrate the present

disclosure, in order to benefit the person having ordinary skill in the art, and can

fully utilize and practice the present disclosure without excessive interpretation.

These examples should not be regarded as limiting the scope of the present

disclosure, but is used to illustrate how to implement the materials and methods

of the present disclosure.

1. Structure of the diarylheptanoid compound of the present disclosure

The diarylheptanoid compound of the present disclosure uses curcumin

(CCM) as a guiding compound to design a curcuminoid diarylheptanoid

compound, which has a structure represented by Formula (1):

O O

Rb Rb' Re Re

Formula (1),

Please refer to the following Table 1, which shows Ra, Rb, Ra', Rb', Ro, Re and

Re' of examples of the diarylheptanoid compound of the present disclosure

Compound 1, Compound 21a, Compound 21b, Compound 22a, Compound 22b,

Compound 23a, Compound 23b, Compound 24a, Compound 24b, Compound 25,

Compound 26, Compound 27, Compound 31, Compound 33, Compound 35a,

Compound 35c, Compound 35d, Compound 35e, Compound 36 and Compound

37.

Table 1

Compound Ra Ra' Rb Rb' Rc Re, Re' 1 OH OH OCH3 OCH3 H H O OH 0 OH 21a OCH3 OCH3 -o-CtCH, OH -o-8CCHOH H H OH 21b OCH3 OCH3 O-CtCH OH H H OH OTOH

22a OCH3 OCH3 -0-C CH5 -0- CH5 H H 2H OH 22b OCH3 OCH3 -0-CtCH, OH OH H H O-OH OCH 23a H H -O-C CH, -O-C CH, H H OFOH

23b H H -O-CtCH - OH H H OH OFOH 33 O CH, OH -0-O-CH3 OH OCH3 OCH3 H H

OH OH 24b CH, OH OH3 OCH3 H H OH

25 OH OH OCH3 OCH3 H H OFOH O OH 26 OC2H5 OC2H5 -O-CtCH OH -O-COCHr OH H H 0 EOH 0FO

31 C2H5 C2H5 -0-C-ECH, -O-C-CH, H C2H5 OF-OH

33 OCH3 OCH3 0 - -CtCH, OCH3 H H OOH OFOH 35a OCH3 OCH3 -0-C-CH, -0-C-CH, CH3 H OOH OFOH

35c OCH3 OCH3 - 0 CCH, - 0 -tCH~ benzyl H OOH OFOH

35d OCH3 OCH3 -CtH ~ OCtH3 propargyl H OF-OH OF-OH

35e OCH3 OCH3 -O-CtCH, -o-8-tCH, allyl H OOH OFOH

36 H H - 0 -CtCH, -0--ICH, CH3 H

IA o OH 0 OH 37 -O-CLCH -O-C-OCH OCH3 OCH3 CH3 H OH LOH

Structures of Compound 21a-((1E,3Z,6E)-3-hydroxy-5-oxohepta- 1,3,6

triene-1,7-diyl)bis(2-methoxy-4,1-phenylene)bis(3-hydroxy-2-hydroxymethyl)-2

methylpropanoate, Compound 35a-((1E,6E)-4,4-dimethyl-3,5-dioxohepta-1, 6

diene-1,7-diyl)bis(2-methoxy-4,1-phenylene)bis(3-hydroxy-2-hydroxymethyl)-2

methylpropanoate, Compound 35d-((1E,6E)-3,5-dioxo-4,4-di(prop-2-yn-1-yl)

hepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-phenylene)bis(3-hydroxy-2

(hydroxymethyl)-2-methylpropanoate), Compound 36-((1E,6E)-4,4-dimethyl-3,5

dioxohepta-1,6-diene-1,7-diyl)bis(4,1-phenylene)bis(3-hydroxy-2

(hydroxymethyl)-2-methylpropanoate), and Compound37-((1E,6E)-4,4-dimethyl

3,5- dioxohepta-1,6-dien-1,7-diyl)bis(2-methoxy-5,1-phenylene)bis(3-hydroxy-2

(hydroxymethyl)-2-methylpropanoate) are shown in Table 2.

Table 2

Compound Structure O OH MeO OMe 0 0 21a HO O 0 OH

HO OH

0 0 MeO OMe 0 0 35a HO o O OH HO OH

0 0

35d HO-_fX I 0I OH HO, OH

0 0

36 HO O 0 OH HOY OH

0 0 0 0

HOH 37 HO HO H3K CH OH

The structure of Compound 21a includes a heptadiene-3,5-dione moiety,

which can be readily interconvertible between the keto form and enol form. The

3- or 5-OH group of the enol form combines with the adjacent 5- or 3-C=O through

hydrogen bonds to stabilize the structure thereof. In the present disclosure, two

methyl functionalities are incorporated onto the 4-position of Compound 21a and

afforded ((1E,6E)-4,4-dimethyl-3,5-dioxohepta-1,6-diene- 1,7-diyl)bis(2

methoxy-4,1-phenylene)bis(3-hydroxy-2-hydroxymethyl)-2-methylpropanoate

(Compound 35a), which is found to possess stable keto form, and is not able to

tautomerization. In addition, in the present disclosure, Compound 35a is used

as a new lead compound and derived into a series of 4,4-dialkyl derivatives

thereof (Compound 35a, Compound 35d, Compound 36 and Compound 37) with

stable keto form.

2. Inhibitory growth effect of the diarylheptanoid compound of the present

disclosure on the EGFR gene mutant NSCLC cells and the EGFR-TKIs resistant

NSCLC cells

The EGFR gene mutant NSCLC cells were treated with the diarylheptanoid

compounds of the present disclosure, and then the cell survival was stained with

crystal violet to measure the IC5ovalues of the diarylheptanoid compounds of the

present disclosure in the EGFR gene mutant NSCLC cells, so as to determine

the growth inhibitory effect of the diarylheptanoid compounds of the present

disclosure on the EGFR gene mutant NSCLC cells.

EGFR gene mutations in lung cancer are mostly found in exons 18-21, which

K are the intracellulartyrosine kinase coding region. The most common mutations include E746-A750del in exon 19 and L858R point mutation in exon 21, which account for about 85%-90% of EGFR gene mutations. Tumor cells with these two mutations are sensitive to the EGFR-TKIs, known as activating mutations.

Secondary mutations may occur in some tumor cells, and the most common

secondary mutation is the T790M mutation in exon 20, which is a drug resistance

mutation. Please refer to Fig. 1A, which shows the analysis result of the

inhibition of the H1650 cells growth by the diarylheptanoid compounds of the

present disclosure. The H1650 cells have the E746-A750del mutation in exon

19 of the EGFR gene, wherein the CCM represents curcumin and the BDMC

represents bisdemethoxycurcumin. The IC50 value of the compound > 16 pM

shows that the H1650 cells have not yet reached 50% cell growth inhibition after

treatment with compounds at concentrations as high as 16 pM, and data in Fig.

1A are represented by mean SD (n = 3).

Fig. 1A shows the IC50 values in the H1650 cells treated with 22

diarylheptanoid compounds (including BDMC) and curcumin for 3 days. 18

diarylheptanoid compounds including Compound 1, Compound 24b, Compound

23b, Compound 33, Compound 25, Compound 35b, Compound 22a, Compound

21a, Compound 22b, Compound 26, Compound 36, Compound 35a, Compound

35c, Compound 27, Compound 31, Compound 37, Compound 35e and

Compound 35d have significantly better inhibitory activity than the parent

compound - curcumin on the growth of the H1650 cells, wherein ** represents p

< 0.05, *** represents p < 0.01, **** represents p < 0.001.

Experimentally, another 7 EGFR-TKIs resistant NSCLC cells were treated

1'7 with Compound 21a, Compound 35a, Compound 35d, Compound 36 or

Compound 37, and then the cell survival assay was performed to measure the

IC5o values of the diarylheptanoid compounds of the present disclosure in the

EGFR-TKIs resistant NSCLC cells, so as to determine the growth inhibitory effect

of the diarylheptanoid compounds of the present disclosure on the EGFR-TKIs

resistant NSCLC cells. The EGFR-TKIs resistant NSCLC cells used in the test

include the H1975 cells, the GR2 cells, the GR5 cells, the GR6 cells, the GR8

cells, the GR9 cells and the GR10 cells, in which the H1975 cells have a point

mutation of L858R in exon 21 and a secondary mutation of T790M in exon 20;

the GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and the

GR10 cells are gefitinib-resistant cell lines obtained after treating gefitinib with the

HCC827 cells as parental cells, while the HCC827 cells and the H1650 cells are

the NSCLC cells with E746-A750del mutation of EGFR exon 19.

Please refer to Fig. 1B, Fig. 1C and Fig. 1D. Fig. 1B shows the analysis

result of the inhibition of the H1975 cells growth by the diarylheptanoid

compounds of the present disclosure. Fig. 1C shows the analysis result of the

inhibition of the GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9

cells and the GR10 cells growth by the diarylheptanoid compounds of the present

disclosure. Fig. 1D shows the percentage changes of the IC5o values after the

GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and the

GR10 cells were treated with Compound 35d and gefitinib respectively. Datain

Fig. 1B and Fig. 1C are presented as mean ±SD (n = 3), while the IC5o value >

16 pM show that the test cells have not yet reached 50% cell growth inhibition

after treatment with compounds at concentrations as high as 16 pM. Gef in Fig.

1D represents gefitinib.

1Q

Fig. 1B shows the measured the IC50 values of the H1975 cells treated with

Compound 21a, Compound 35a, Compound 35d, Compound 36, Compound 37

and curcumin for 3 days, respectively. The results showed that Compound 21a,

Compound 35a, Compound 35d, Compound 36 and Compound 37 of the present

disclosure had significantly better inhibitory activity than curcumin on the growth

of the H1975 cells.

Fig. 1C shows the measured the IC5o values of the GR2 cells, the GR5 cells,

the GR6 cells, the GR8 cells, the GR9 cells and the GR10 cells after treatment

with Compound 21a, Compound 35a, Compound 35d, Compound 36, Compound

37 and curcumin for 3 days, respectively. The results show that Compound 21a,

Compound 35a, Compound 35d, Compound 36 and Compound 37 of the present

disclosure have significantly better inhibitory activity than curcumin on the growth

of the GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells, the GR9 cells and

the GR10 cells.

The results in Fig. 1B and Fig. 1C show that all EGFR-TKIs resistant NSCLC

cells are more sensitive to Compound 21a, Compound 35a, Compound 35d,

Compound 36 and Compound 37 than curcumin, and can achieve 50% inhibition

of cell growth at significantly lower concentrations.

Fig. 1D shows that the analysis results obtained by comparing the IC5o

values measured by the GR2 cells, the GR5 cells, the GR6 cells, the GR8 cells,

the GR9 cells and the GR10 cells with the IC5o value measured by the HCC827

cells, in which the HCC827 cells, the GR2 cells, the GR5 cells, the GR6 cells, the

GR8 cells, the GR9 cells and the GR10 cells were treated with Compound 35d

and gefitinib for 3 days and then measured the IC5o values. The results in Fig.

1D show that all gefitinib-resistant cell lines are indirectly sensitive to Compound

35d. All gefitinib-resistant cell lines were >200-fold more resistant to gefitinib

(Gef) than their parental cells, the HCC827 cells.

3. Anticancer activity of Compound 35d against the GR6 tumor, the GR8

tumor and the HCC827 tumor

In order to verify the anticancer effect of the diarylheptanoid compound of

the present disclosure in vivo, the GR6 tumor mouse model, the GR8 tumor

mouse model and the HCC827 tumor mouse model of xenotransplantation were

first established, and the GR6 tumor mice, the GR8 tumor mice, and the HCC827

tumor mice were treated with 100 mg/kg of Compound 35d daily for 35 days, and

the tumor size and body weight of the GR6 tumor mice, the GR8 tumor mice, and

the HCC827 tumor mice were recorded.

Please refer to Fig. 2A to Fig. 2D, Fig. 2A shows the analysis result of the

inhibition of tumor growth in the GR6 tumor mice by treating Compound 35d alone,

Fig. 2B shows the analysis result of the inhibition of tumor growth in the GR8

tumor mice by treating Compound 35d alone, Fig. 2C shows the analysis result

of the inhibition of tumor growth in the HCC827 tumor mice by treating Compound

35d alone, and Fig. 2D shows the statistical chart of body weight changes in the

tumor mice by treating Compound 35d alone, wherein data in Fig. 2A to Fig. 2C

are presented as mean ±SEM (n = 10).

The results in Fig. 2A to Fig. 2C show that Compound 35d significantly

inhibits the tumor growth of the GR6 tumor mice and the GR8 tumor mice, but

has less significant effect on inhibiting the tumor growth of the HCC827 tumor

mice. However, the results in Fig. 2D show that the body weight of the tumor mice did not decrease significantly after being treated with Compound 35d for more than 1 month.

4. Combined treatment of Compound 35d and osimertinib inhibits the re

progression of the GR6 tumor

EGFR-TKIs are currently the standard treatment for NSCLC patients with the

EGFR gene mutation. To further test whether the combined use of the

diarylheptanoid compound of the present disclosure and known drug can

enhance the therapeutic effect of NSCLC, the GR6 tumor mice were divided into

4 groups. One group received 100 mg/kg Compound 35d treatment per day

io (represented as 35d), another group received 1 mg/kg osimertinib treatment per

day (represented as Osi), still another group received the combined treatment of

100 mg/kg Compound 35d and 1 mg/kg osimertinib per day (represented as

35d+Osi), and the other group was the control group without drug treatment.

Please refer to Fig. 3A and Fig. 3B, Fig. 3A shows the analysis result of the

inhibition of tumor re-progression in the GR6 tumor mice by treating the combined

treatment of Compound 35d and osimertinib, and Fig. 3B shows the statistical

chart of body weight changes in the GR6 tumor mice by treating the combined

treatment of Compound 35d and osimertinib, wherein data in Fig. 3A and Fig. 3B

are represented by mean ±SEM (n = 10).

The results in Fig. 3A show that although the tumor size of the GR6 tumor

mice in the Osi group was initially reduced by osimertinib treatment, the treated

tumors subsequently re-growth, indicating that the tumors of the GR6 tumor mice

have EGFR-TKIs resistance. However, no matter in the group treated with

Compound 35d alone or in the group treated with the combined treatment of

'1

Compound 35d and osimertinib, tumor re-progression in the GR6 tumor mice was

significantly inhibited. And the results in Fig. 3B show that neither the single

treatment of Compound 35d nor the combined treatment of Compound 35d and

osimertinib can significantly reduce the body weight of mice.

To sum up, the present disclosure provides a new use of the pharmaceutical

composition, which can be used to manufacture a drug for treating lung cancer.

The pharmaceutical composition includes the diarylheptanoid compound or the

pharmaceutically acceptable salt thereof, which can inhibit the growth of the

EGFR gene mutant NSCLC cells and the EGFR-TKIs resistant NSCLC cells, so

io that can be used to manufacture the drug for treating lung cancer. Moreover,

the pharmaceutical composition can have a synergistic effect when used in

combination with EGFR-TKIs, which can increase the effectiveness of treating

lung cancer, especially for the treatment of lung cancer with the EGFR gene

mutation and EGFR-TKIs resistant, and has the potential to be used in the

medical and health care market.

Although the present disclosure has been described as above by way of

embodiments, it is not intended to limit the present disclosure. Person having

skilled in the art can make various changes and modifications without departing

from the spirit and scope of the present disclosure. Therefore, the scope of

protection of the present disclosure should be defined by the scope of the

appended patent application.

Throughout this specification, unless the context requires otherwise, the

word "comprise", and any variations thereof such as "comprises" or "comprising",

are to be interpreted in a non-exhaustive sense.

Claims

1. Use of a pharmaceutical composition in the manufacture of a medicament for the treatment of a non-small cell lung cancer, wherein the pharmaceutical composition comprises a diarylheptanoid compound or a pharmaceutically acceptable salt thereof, and the diarylheptanoid compound is selected from:

MeO 00 OMe MeO OMe O 0 0 0 HO o -0 OH HO 0 0 OH

HO OH HO OH

0 0

0 0 HO 0 0 OH HO OH and 0 0 0 0 HO'_ O: o, _ 0 _1- .OH HO HOH

2. A use as claimed in claim 1, wherein the diarylheptanoid compound is 0 0 MeO OMe 0! 0 HO 0 0 OH

HO OH

3. A use as claimed in claim 1 or 2, wherein the pharmaceutical composition further comprises an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI).

4. A use as claimed in claim 3, wherein the EGFR-TKI is osimertinib, gefitinib, erlotinib or afatinib.

5. A use as claimed in claim 1 or 2, wherein the non-small cell lung cancer is resistant to an epidermal growth factor receptor-tyrosine kinase inhibitor.

6. A method of treating a non-small cell lung cancer, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a diarylheptanoid compound or a pharmaceutically acceptable salt thereof, wherein the diarylheptanoid compound is selected from:

MeO N OMe MeO OMe 0 0 0 0 HO 0 O OH HO O0 O0 OH

HO OH HO OH

0 0

0 O HO O 0 OH HO OH and 0 0 0 0 0 HO N OH

HO OH

HCO QCH 3